مجله بیوتکنولوژی کشاورزی
سال پانزدهم شماره 1 (پیاپی 50، بهار 1402)

Abstract
The current study was undertaken to investigate if there is a relationship between metabolism of ethylene and heavy polyamines (PAs) under cold stress in cold-tolerant and cold-sensitive chickpea (Cicer arietinum L.) genotypes.
 
In this research, content of ethylene, heavy polyamines (spermidine (Spd) and spermine (Spm)), activities of PAs degradation pathway enzymes (polyamine oxidase (PAO) and diamine oxidase (DAO)), hydrogen peroxide (H2O2) and relative expression of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane 1-carboxylic acid oxidase (ACO) genes in cold-tolerant (Sel 96th11439) and cold-sensitive (ILC 533) chickpea (Cicer arietinum L.) genotypes during the first and sixth days of cold stress at 4 °C compared to control condition as a factorial experiment in a Completely Randomized Design were investigated.
 
During cold stress, both genotypes showed a significant increase in Spd and Spm content (66.66 and 96.23%). Ethylene production was declined in cold-sensitive genotype under cold stress compared to control conditions (up to 26.08%) while in the cold-tolerant genotype, the unique ethylene peak in early response (on the first day of stress) comared to control conditions (15.62%) was closely related to increased heavy polyamine accumulation. In the tolerant genotype, the increase in polyamine oxidase (PAO) and diamine oxidase (DAO) activity in early responses, (By 2.6- and 3.01-fold, respectively) was related to the increase in ethylene biosynthesis, as well as a concomitant increase in heavy polyamine (Spd & Spm) content by cold stress. In the tolerant genotype, the relative expression of ACS and ACO genes, after a significant increase on the first day of cold stress (5.2- and 4.03-fold, respectively), showed a significant decrease on the sixth day of the stress compared to the control plants, while a continuous decreasing trend (35-and 21.7-fold, respectively) was observed in the sensitive genotype compared to the control condition.
 
Findings of this research suggest that ethylene is intimately involved in improvement of cold stress tolerance through activation of a complex pathway of signalling by H2O2 that is polyamine catabolism-dependent.

Lily is known as the third main bulbous flower in the world that used for cut flowers and pots. Bulblet as a staple procedure is known for propagation of this plant. Due to the high performance of in vitro bulblets regeneration, the use of this method can reduce the costs production of this plant and also reduction of water and soil pollution due to minimizing the field area for ex vitro propagation. The size of lily bulblets produced in vitro is smaller than ex vitro propagated bulblets. For this purpose, several concentrations of phosphorus, potassium and calcium were used to investigate their role on lily bulblet growth under in vitro condition.
 
In this experiment, ‘Santander’ cultivar was used for bulblet growth of lily. For this purpose, the effect of five strength (0, 0.5, 1, 2, 3) of macro elements (phosphorus, potassium and calcium) based on MS medium on bulblet regeneration percentage, bulblet number, bulblet fresh weight, scale explant fresh weight, and fresh weight of leaf and root was surveyed.
 
The results of the experiment clearly showed the effect of all three elements on the growth of bulblet, so that the limitation of any of the elements (0 strength) caused serious disorders in all explants.  With increasing the concentration of the studied elements, the bulblet regeneration percentage increased significantly. The maximum in vitro bulblet regeneration percentage (95.3 %) was found in explants grown on medium supplemented with double-strength of phosphorous. All three macroelementsnotably phosphorus played a major role on lily bulblets growth. According to results, application of double-strength of phosphorus caused the highest number of bulblet compared to test with 11.6. A considerable decrement in the fresh weight of leaves (16 g) and roots (10 g) was observed in the absence of calcium in the culture medium.
 
In general, phosphorus and calcium play a vital role in the regeneration of lily bulblets in vitro, so that on the one hand, double-strength of phosphorus had the highest amount of bulblet regeneration and fresh weight of the bulblets and on the other hand, shortage of calcium had the lowest amount in all parameters. So it is recommended to increase the concentrations of above mentioned macroelementsfor obtaining bigger bulblets.

Objective:

Salinity stress is one of the most important environmental stresses that significantlyhas negative effects on plant growth and crop production all over the world. Among cereal crops, barley (Hordeum vulgar L.) is the most tolerate cereal to abiotic stresses, especially to salinity stress. In the present study, to evaluate the molecular response of some promising genotypes of barley to salinity stress, the expression patterns of APX, GPX, SOD, Rbohf1, and Rbohf2 genes were assessed under two control and salt stress conditions.

Materials and methods:

In this study, the effect of salt treatment (200 mM NaCl) on relative expression patterns of APX, GPX, SOD, Rbohf1, and Rbohf2 genes in a set of six promising genotypes of barley along with a local cultivar as the reference genotype (cv. Mehr) was evaluated. The experiment was conducted in a randomized complete block design in two environments (normal and saline stress) under controlled glasshouse conditions using a hydroponic system. After seedling establishment and applying stress treatment (21-days), plants were subjected to sampling and the relative expression for targeted genes was estimated as proposed by Pfaffl (2001).

Results:

According to the results, a significant difference was observed between control and salt treatments, as well as, among tested barley genotype in terms of all investigated genes. Salt treatment significantly increased expression of Rbohf1, APX, GPX, Rbohf2, and SOD by 6.80, 4.51, 4.26, 2.76, and 2.72-fold compared to control treatment, respectively. A comparison of the expression patterns of studied genes revealed that promising genotypes G6 and G7 showed better tolerance against oxidative stress induced by salt treatment and revealed an acceptable tolerance to salinity stress.

Conclusions:

Our results revealed that the genotypes G6 and G7 due to their genetic background have a high potential to the regulation of their antioxidant system. Hence, these genotypes can be used as ideal candidates for supplementary studies and even participate in adaptability and stability trials to release as commercial cultivars tolerant to salt stress.

Conventional gene delivery to plant cells approaches have limitations such as narrow host range in Agrobacterium-mediated gene delivery method, removal of cell wall using polyethylene glycol and electroporation methods, and the higher cell damage in the use of gene gun. Recently, nanotechnology-based gene delivery methods have been developed for plant genetic transformation, and this nanostrategy shown the efficiency of gene transfer and biocompatibility and the target DNA protection.
Superparamagnetic iron oxide nanoparticles (SPIONs) were fabricated via green route using Catharanthus roseus leave aqueous extract. SPIONs nanoparticles and carboxylated single-walled carbon nanotubes (SWCNTs-COOH) were functionalized with polyethyleneimine (PEI). For gene delivery to Catharanthus roseus leave, the pDNA@SPIONs and pDNA@SWCNTs nanocarriers were prepared by pDNA loading on the surface of cationic nanoparticles. Two methods were used to accelerate and increase the pass of the nanocarrier through the plant cells, immersion in nanocarrier suspension and infiltration by syringe. Tracking the mGFP5 protein fluorescent signal and RT-PCR reaction was evaluated to affirm mgfp5 gene delivery and its expression in plant cells.
The color change of the iron chloride salts solution, the absorption of the synthesized nanoparticles to a magnet, while the iron chloride salts do not have this property, and the results of the performed analysis confirmed the green synthesis of SPIONs nanoparticles. The functionalized nanoaprticles with PEI shown good ability to interact with DNA and effectively DNA protection against nuclease enzymes degradation. The mGFP5 protein fluorescent signal confirmed the gene delivery to plant cells ability of pDNA@SPIONs and pDNA@SWCNTs nanocarriers.
So the application of nanobiotechnology and nanocarriers for gene delivery to plant cells can be promising for plant genetic engineering. By nanocarriers, it is possible efficient gene delivery to almost all plant species regardless dicotyledonous or monocotyledonous through a method with features such as simple and low cost, elimination of Agrobacterium and its limitations, and without the specialized laboratory equipment.

The camelthorn (Alhagi maurorum) is one of the compatible plant species with arid and semi-arid regions that is important for forage production, soil protection and medicine. This research was carried out in order to investigate the genetic diversity of camelthorn ecotypes, to determine the similarities and differences between ecotypes and to recognize the genetic structure of different camelthorn ecotypes using ISSR and SCoT markers.
 
The 22 ecotypes from different regions of North, Razavi and South Khorasan provinces were studied. The DNA was extracted by the CTAB method. The 12 ISSR and 18 SCoT primers were used in the molecular analysis.
 
The polymorphism percentages in the two markers were 99.42% and 95.42%, respectively. In the ISSR and SCoT markers, the IS5, IS8, IS10 and S2, S4, S6, S7, and S10 primers amplified the highest number of bands, respectively, and the IS1, IS2, IS3, IS6 and S1 amplified the lowest bands, respectively. In the ISSR and SCoT markers, the IS6 (0.44) and S12 (0.44) primers had the highest PIC value, respectively, and the IS4 (0.09) and S1(0.04) primers had the lowest PIC value. In the ISSR marker, the IS6 primer had the highest and the IS2, and IS10 primers had the lowest Nei genetic diversity index and Shannon information index, respectively. In the SCoT marker, the S14 primer had the highest, and the S1 primer had the lowest Nei genetic diversity index and Shannon information index, respectively. The cluster analysis classified the ecotypes into three groups. The molecular analysis of variance showed that the 9%, 14% and 91%, 86% of the total genetic variation were related to the within and between groups in ISSR and SCoT markers, respectively.
 
This study demonstrated the accuracy of the ISSR and SCoT markers in identifying high levels of polymorphism and was appropriate for investigating the genetic diversity amongst camelthorn ecotypes. The IS6 and S12, and S14 primers were recognized as the best primers in this study, and it is suggested that these primers be considered in future studies. In total, the results showed that the diversity within populations was higher than the diversity between populations, which indicates that the geographical distribution does not follow of genetic diversity in the camelthorn plant.

Iran is one of the few countries where the origin of pomegranate (Punica granatum L.) in the world. About half of the pomegranate genotypes are endangered. Despite the similarity between some genotypes in terms of appearance, there are differences between anthocyanins, phytochemicals, antioxidants, etc., of them that are very much affected by the environment (especially stress) and require DNA barcoding. For this reason, more accurate molecular studies, especially DNA identification, are necessary to aid in classification, identification of the required genotype, non-incorrect naming of genotypes, and identification of cultivars. By performing this research, it is possible to reduce the volume of genotypes and eliminate duplicate and similar genotypes in Saveh pomegranate collection to reduce their maintenance and management costs. Also used to select the best plant barcode to identify, differentiate and determine the diversity of genotypes for use in breeding programs.
In this study, 58 genotypes in Saveh pomegranate collection were examined by ITS barcode region. After receiving the sequences, first all the sequences blast in the NCBI site for the accuracy of the desired area and after sure of the desired plant area (pomegranate), the quality of the sequences was measured with Chromas software and in addition to deleting the M13 sequence, the beginning and end sequences and poor-quality sequences were deleted. Then, for multiple alignments by clustalW method, bioinformatics analysis was performed using MEGA software.
The results showed that the success rate of propagation in this area by PCR was 79%. Also, the success rate of sequencing in this area was 74.68%. The GC content of this area was 64.23%. The lowest genetic distance for this region (0.005) was between the genotypes of Malls Tabas with Chatroud Shirin and the Torsh Dorag Rafsanjan with Globland Bafgh and the highest genetic distance (5.314) was between the genotypes of Gol Magasi Taft with Dane Siyah Ardestan, Poost Ghermez Zanjan, Malas Paveh, Peyvndi Ashkzar and Dane Ghermez Zavareh genotypes. The results of phylogenetic tree also showed that wild genotypes of Tamin Khash, Domezeh Bagh Malek Izeh and Dorag Malas Bajestan and Gol Magasi Taft were each in separate groups and other genotypes were in other groups.
In general, Ardestan, Khash, Gol Magasi Taft, Malas Peyvandi Ashkzar, Zanjan, Paveh, Zavareh and Ravar genotypes had the highest genetic diversity and distance with other genotypes that according to other characteristics of genotypes, they can be used as parents for breeding programs. Also, considering that this region, unlike other regions, carries both paternal and maternal genes and due to the facilitation of reproduction and the success of their sequences, was identified as a suitable region to show genetic diversity between genotypes which can be used in future research.

This research aimed to identify Fusariums belonging to Elegans and Martiella-Ventricosum sections related to cucurbit plants in Tehran province and to evaluate the ability of ISSR markers to investigate the kinship relationships of these groups of Fusariums.
 
In this research, five ISSR primers (AG)8C, (AG)8G, (AC)8YA, (ATG)6 and BHB(GA)7 were selected. Fungal isolates identified based on morphological and microscopic characteristics. For ISSR markers, the statistics of polymorphism information content (PIC), Marker Index (MI) and Effective Multiplex Ratio (EMR) were calculated. Relationships and similarity between Fusarium isolates were analyzed using NTSYS-PC V2.02.
 
Out of 96 Fusarium isolates, 48 isolates were identified as Fusarium solani, 25 isolates as Fusarium oxysporum and 23 isolates as Fusarium redolens. A total of 408 bands were obtained from ISSR amplifications. 130 different bands were produced, of which 83 were polymorphic and 47 were monomorphic. The size of PCR fragments produced varied from 273 to 2428 bp. The polymorphism percentage, ranged from the highest (73%) for (ATG)6 to the lowest (56%) for (AG)8C. Primers (ATG)6 and BHB(GA)7 performed better in differentiation Elegans and Martiella-Ventricosum sections from each other. Primers (AG)8C with 26 gene loci, and BHB(GA)7 with 25 gene loci, produced the most bands in Elegans isolates and Martiella-Ventricosum isolates, respectively.
 
The results of this research showed that there was a high genetic diversity among the investigated Fusarium isolates and that the ISSR markers are efficient markers to reveal the molecular relationships between Fusarium populations.

Objective:

Pulicaria gnaphalodes (Vent.) Boiss, known as Kak-Kosh, is a desert-adapted species that has been widely distributed throughout Iran. This plant is a source of flavonoids and terpenes that are used in traditional medicine. Considering the medicinal importance of this plant, the aim of this research was to investigate the effectiveness of molecular and morphological markers to separate the populations.

Materials and methods:

In this research, 20 populations of this species were collected from Kerman, Fars, Yazd and Hormozgan provinces and their genetic diversity was analyzed using 10 morphological traits and 25 ISSR primers. Sampling for genetic and morphological studies were carried out in spring and late summer, respectively.

Results:

A total of 150 ISSR marker fragments were scored and 139 bands were found to be polymorphic [the percentage of polymorphic bands (PPB): 92.67%]. The average number of effective alleles (Ne) and expected Heterozygosity (uHe) for the amplification products were 1.470 and 0.288 respectively. Both Principal Coordinates Analysis (PCoA) and UPGMA cluster analysis supported the clustering of all the populations into two groups. Small cluster contained populations 15, 18 and 19 and other populations were placed in a large Cluster. A weak relationship was observed between genetic diversity and GIS data. The Factor Analysis (FA) results for morphological traits detected three principal components with Eigen value greater than one (reproductive, leaf area, and vegetative components), which explained 69.24% of the total variability. These three components explained 28.77, 21.05 and 19.42 percentage of variability respectively. The FA analysis separated a group from the other populations.

Conclusions:

In the present study, high levels of genetic and morphologic diversity were found between P. gnaphalodes populations. So that geographical separations have a weak effect on genetic diversity of populations. It seems that some probable factors such as perianality, self-incompatibility, pollination system, seed dispersal by wind, and gene flow could support achieving the population in a high level of genetic diversity.

APETALA1 (AP1) gene plays an important role in promoting transition from vegetative to reproductive phase and determining the flower meristem identity. This research was conducted to investigate the AP1 relative expression in the black mustard plant (Brassica nigra L.). Also, the effect of hydrogen sulfide on flowering and its relative expression was investigated.
Total RNA was extracted from the collected samples and used to make cDNA. Specific primers were designed and used for RT-PCR reaction based on the sequence alignment of AP1 homologous genes in plants of the same family. The relative expression of AP1 was studied in the reproductive phase in different organs such as root, stem, leaf, flower bud, sepal, petal, stamen and pistil. In addition, the effect of NaHS on flowering and relative gene expression in different developmental stages in vegetative shoots, generative shoots and flower buds was investigated. The intensity of expression was measured with ImageJ software and the data were compared with ANOVA statistical analysis and Duncan's test with a confidence factor of 95%.
 
 
Studies of the relative expression of AP1 gene in different organs of the black mustard in the generative phase indicated that this gene is expressed in flower bud, sepal, petal and leaf, but no expression was observed in the root, stem, stamen and pistil. Also, its relative expression in samples treated with NaHS compared with the control group showed that the beginning of AP1 gene expression and consequently the transition to flowering is faster and earlier in plants treated with hydrogen sulfide. Thus, the treated plants had a shorter life cycle and flowered 8 days earlier. The comparison of its expression in vegetative shoots, reproductive shoots and flower buds did not show any significant difference in the same developmental stages, but expression higher levels was observed at the same sampling times in the treated samples compared with controls. No expression was observed in the control samples at the periods that the treated samples showed high expression and were in generative phase and flowering.
The relative expression of AP1 gene during developmental stages and its increase in the reproductive stage can confirm its role in the flowering process. NaHS induced flowering and in a 35-day life cycle, treated plants entered the reproductive phase 8 days earlier. Therefore, NaHS treatment, by shortening the vegetative period, stimulated the precocious expression of AP1 gene and consequently its flowering.

Plant diseases are one of the major limitations in agricultural production. Citrus blast disease is one of the most common diseases in citrus producing regions of the world, with the exception of tropical regions. Plants respond to bacterial pathogens by activating genes encoding defense proteins. One of the defense mechanisms of plants against pathogens is the accumulation of Pathogenesis related proteins associated. The aim of this study was to evaluate the expression of several defense genes against Pseudomonas viridiflava after treatment of seedlings with Salicylic acid and Jasmonic acid.

The expression levels of PR1, PR2, PR3, PR4, PR5, POX, LOX pathogenesis related and PAL genes in the early stages of citrus blast disease in Citrus aurantium were assessed using RT-PCR. In this study, UBI gene was used as a housekeeping gene. The total RNA of the samples was extracted and after cDNA synthesis, the expression level changes of the declared genes were calculated and measured using the formula 2 - (ΔΔCT).

Increased expression of PR1, PR2, PR3, PR4, PR5, POX, LOX Pathogenesis related and PAL genes in the early stages of citrus blast disease in sour orange mostly occurred at 24 and 48 hours after challenging with the citrus blast causal agent P. viridiflava.

The results of this study was showed that salicylic acid treatment had a greater effect on the expression of PR1, PR2, PAL, POX and PR5 genes than jasmonic acid and jasmonic acid treatment was showed a greater effect on the induction of PR4, PR3 and LOX genes than salicylic acid. Furthermore, the expression of PR1, PR2, LOX, PR4, PAL and PR5 genes peaked in 24 hours after infection in sour orange plant by both treatments and it seems that these mentioned inducer of resistance genes involved in citrus blast disease resistance in host plant

Objective:

Cannabis seeds have high amounts of crude protein and energy, so they can better resist the microbial digestion of the rumen and pass through the intact rumen to a greater extent and be digested and absorbed in the intestine. This means they can improve livestock performance. Therefore, they can be used for this purpose. On the other hand, DLK1 gene plays an important role in controlling cell differentiation processes, including muscle and nerve differentiation, adipogenesis, hematopoiesis, and bone differentiation throughout fetal and adult life. Therefore, the aim of this study was to investigate the effect of cannabis seeds on DLK1 gene expression in the heart tissue of Kermani lambs.

Materials and Methods:

To carry out this research, 12 six-month-old Kermani lambs of equal weight were used in a completely randomized design in three experimental groups.After slaughter, sampling of heart tissue (with 3 repeats) was done from both groups.The samples were quickly placed in liquid nitrogen and then stored in a -80 freezer.Total RNA was extracted using a standard kit.The quality and quantity of extracted RNA was evaluated.cDNA synthesis kit of Fermentase Company was used for cDNA synthesis.Real time PCR reaction using Syber Green method was used to check the relative level of gene expression.To analyze the data obtained from real time PCR, the method of Pfaffl et al. was used.

Results:

The ratio of A260/A280 for the extracted RNAs was equal to 1.9-1.8, which indicates their appropriate and desirable quality. The results of real time PCR curves and electrophoresis of PCR products on agarose gel showed that DLK1 gene is expressed in heart tissue. The results of the present study also showed that feeding cannabis at the level of 1% significantly (P<0.05) increases the expression of DLK1 gene in the heart tissue by 4.74 times compared to the control group.

Conclusions:

Based on the results of the present study, it can be concluded that cannabis can be used in the diet of sheep to improve the structure of the heart through positive effects on the expression of the DLK1 gene. In addition, the interesting results of the present study in response to the use of Cannabis seeds in the diet of sheep open a new path for wider research in this field.

Rosmarinic acid (RA), an anticancer, antiallergic, and antimicrobial agent, is a secondary metabolite in many plants of the family Lamiaceae including M. officinalis L. The application of nanoparticles (NPs) as a novel elicitor for the biosynthesis of bioactive compounds shown that the NPs could affect the secondary metabolites in plants by eliciting the expression of biosynthetic pathway genes. The present paper aimed to assess the effect of Magnetic Iron Oxide Nanoparticles (MIONPs) in comparison with their dissolved counterpart.

Foliar application of different concentrations: including 5, 10, 25, and 50 mg/L Fe of ferric chloride and MIONPs on the plant leaves was performed. The relative mRNA levels of TAT, RAS, and HPPR were evaluated with quantitative real-time PCR (qRT-PCR) and compared between treated and untreated samples.

This study showed the positive effects of ferric chloride and MIONPs on the expression of genes involved in the biosynthesis pathway of RA. The highest expression level of TAT, HPPR, and RAS genes was observed in plants treated with MIONPs at the concentration of 25 mg/L and the expression of genes decreased as the concentration increased to 50 mg/L. However, the genes expression was still higher compared to the control plant.

The results showed that the exposure of plants to ferric chloride and magnetic iron oxide nanoparticles led to an increase in the expression of the genes under study compared to control samples. However, the application of nano-scale iron particles had more effect than ferric chloride on the expression levels. An increase in the expression of genes involved in the biosynthesis pathway of RA through treatment with MIONPs provides an opportunity for induction of synthesis and accumulation of RA. Therefore, we hope to be able to enhance the production of RA in M. officinalis L. by using these NPs.